1. Field of the Invention
This invention relates to a microorganism that is a mutant bacterial strain of the species Actinosynnema pretiosum, designated strain PF4-4 (ATCC PTA-3921), being capable of producing maytansinoid ansamitocins such as ansamitocin P-3 in improved yield compared to previous known strains, and to methods of producing such maytansinoid ansamitocins from said strain PF4-4.
2. Background of the Invention
Bacteria of the species Actinosynnema pretiosum produce cytotoxic maytansinoid antibiotics (Higashide et al. Nature 270, 721-722, 1977). Bacteria of this species were originally classified and deposited as Nocardia sp., however subsequent characterization demonstrating the absence of any mycolic acids, cell wall type III/C (meso-diaminopimelic acid and no diagnostically important carbohydrates), a lack of sporangia, and the formation of motile elements, indicated that these strains are members of the genus Actinosynnema (Hasegawa, et al. xe2x80x9cMotile Actinomycetes: Actinosynnema pretosium subsp. pretosium sp nov., subsp. nov., and Actinosynnema pretosium subsp. auranticum susp. nov.xe2x80x9d Int. J. System. Bacteriol. 33(2):314-320, 1983).
The bacterially produced maytansinoids are called ansamitocins, and comprise a group of antitumor benzenoid ansamycin antibiotics that are distinguished from one another by their substitutions at the C-3 and C-14 positions, as shown by substituents R and R1 of formula (I).
Several strains of Actinosynnema have been deposited, such as ATCC 31565, Actinosynnema pretiosum subsp. auranticum. The metabolic, physiological and maytansinoid-producing properties of ATCC 31565 are described in U.S. Pat. Nos. 4,331,598 and 4,450,234 to Hasegawa et al., issued on May 25, 1982, and May 22, 1984, respectively. ATCC 31565 is a gram-positive bacterium that is capable of growth on a wide range of carbon sources and which produces principally a mixture of maytansinoids and of C-14-hydroxymethyl substituted be harvested from the growth medium in low yield.
Maytansinoids were originally isolated from African plants (Kupchan et al. J. Amer. Chem. Soc. 94, 5294-5295, 1972). Production of maytansinoids from such sources was difficult because they were present in very small amounts. A maytansinoid-producing microorganism was subsequently isolated from sedge blades, which was classified as a new strain of the genus Nocardia, Nocardia sp. strain No.C-15003 (N-1). This strain was deposited as ATCC 31281, and is disclosed in U.S. Pat. No. 4,137,230 to Hashimoto et al., issued Jan. 30, 1979, and U.S. Pat. No. 4,162,940 to Higashide et al., issued Jul. 31, 1979. Purification of maytansinoids from this bacterium requires fewer steps and results in increased yield compared to purification from plant sources.
A second maytansinoid-producing strain was isolated from sedge blades, named Nocardia sp. Strain No. C-14482 (N-1001), deposited as ATCC 31309. This strain is disclosed in U.S. Pat. No. 4,292,309 to Higashide et al., issued Sep. 29, 1981.
A third strain was derived from ATCC 31309, designated C-14482, by a process of mutagenesis. This third strain was named Nocardia sp. No. N-1231, and was deposited as ATCC 31565. U.S. Pat. Nos. 4,331,598 to Hasegawa et al., issued May 25, 1982, and 4,450,234 to Hasegawa et al., issued May 22, 1984, disclose ATCC 31565.
All three of the above-mentioned Nocardia sp. strains produce maytansinoids called ansamitocins in small amounts. Thus, methods have been disclosed for the production of ansamitocin P-3 using Nocardia sp. Strain No.C-15003 (see, U.S. Pat. No. 4,356,265, to Hatano et al., issued Oct. 26, 1982; and Hatano et al. xe2x80x9cSelective accumulation of ansamitocins P-2, P-3 and P-4, and biosynthetic origins of their acyl moietiesxe2x80x9d Agric. Biol. Chem. 48, 1721-1729, 1984). According to these methods, relatively small amounts of the desired product, ansamitocin P-3, are obtained, with yields of about 100 mg/L of fermentation broth.
Maytansinoids have potent cytotoxic activity and have demonstrated strong anti-tumor activity when delivered in conjugate form with a cell-binding agent. For example, U.S. Pat. No. 5,208,020 to Chari et al., issued May 4, 1993, discloses a cytotoxic agent comprising one or more maytansinoids linked to a cell targeting agent such as an antibody, whereby the maytansinoid is directed toward killing selected cell populations through the specific cell-binding agent. Likewise, U.S. Pat. No. 5,416,064, also to Chari at el., issued May 16, 1995, discloses new maytansinoids that are attached to cell-binding agents through cleavable disulfide linkages, whereby the maytansinoid is released intracellularly. These conjugates have pharmaceutical potential for the treatment of various cancers.
Because of the many therapeutic uses of maytansinoids, there exists a need for new strains of bacteria that are capable of producing ansamitocins in improved yield and in sufficient quantities to facilitate commercial development, for example, of such anti-cancer agents as described above and disclosed in U.S. Pat. Nos. 5,208,020 and 5,416,064. The present invention fulfills this need and more, as will be apparent to one of skill in the art upon reading the following disclosure and examples.
The present invention provides a bacterial strain, deposited as ATCC PTA-3921, also termed herein xe2x80x9cPF4-4,xe2x80x9d that produces increased amounts of maytansinoids. PF4-4 was obtained by mutation from parent strain N-1231 (ATCC 31565) using ultraviolet light (UV light), 1-methyl-3-nitro-1-nitroso-guanidine (MNNG), and selection for enhanced maytansinoid production.
Thus, in a first embodiment, the invention comprises a mutated bacterial strain (PF4-4) of the species Actinosynnema pretiosum that produces much higher quantities of ansamitocins than the parental strain.
This embodiment of the invention is capable of producing more than 500 mg/L of ansamitocin P-3, which is a yield improvement of 5- to 10-fold compared to the parental strain.
This embodiment is further capable of producing substantial amounts of other ansamitocin species, for example ansamitocins P-2 and P-4. Furthermore, the relative amounts of specific ansamitocin species that are produced by this embodiment of the invention are capable of rational manipulation through the choice of carbon source used to support growth.
This embodiment is capable of growth upon a wide variety of carbon sources, and, with the exception of its capability to produce increased amounts of maytansinoids, is substantially similar to the parental strain (ATCC 31565) with respect to its morphology, physical and metabolic characteristics.
Thus, one object of the present invention is to provide a bacterial strain that is capable of enhanced maytansinoid production, whereby such maytansinoids are highly cytotoxic and can be used as therapeutic agents, for example in the form of a conjugate with a cell-specific component, in the treatment of many diseases, including cancer.
A second object of the invention is to provide a bacterial strain that is capable of enhanced maytansinoid production such that maytansinoid may be produced in sufficient quantities to facilitate commercial development of said therapeutic agents. to A third object is to provide a method for the production of maytansinoid ansamitocins from strain PF4-4 by culturing said strain in a growth medium comprising a suitable carbon source. The proportions of maytansinoid ansamitocins produced by this method may be predetermined by the choice of carbon source.